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Generation of Stably Transfected Mammalian Cell Lines as Fluorescent Screening Assay for NF- B Activation-Dependent Gene Expression

Radiation Biology, Institute of Aerospace Medicine, DLR, Linder Höhe, D-51170 Köln, Germany, christine.hellweg{at}dlr.de

Christa Baumstark-Khan

Radiation Biology, Institute of Aerospace Medicine, DLR, Linder Höhe, D-51170 Köln, Germany

Gerda Horneck

Radiation Biology, Institute of Aerospace Medicine, DLR, Linder Höhe, D-51170 Köln, Germany

Cellular stress protection responses lead to increased transcription of several genes via modulation of transcription factors. Activation of the Nuclear Factor B (NF-B) pathway as a possible antiapoptotic route represents one important cellular stress response. To identify conditions that are capable of modifying this pathway, a screening assay for detection of NF-B-dependent gene activation using the reporter protein Enhanced Green Fluorescent Protein (EGFP) and its destabilized variant (d2EGFP) was developed. Human Embryonic Kidney (HEK/293) cells were stably transfected with a vector carrying EGFP or d2EGFP under control of a synthetic promoter containing 4 copies of the NF-B response element. Treatment with tumor necrosis factor (TNF-) gave rise to substantial EGFP/d2EGFP expression in up to 90% of the cells and was therefore used to screen different stably transfected clones for induction of NF-B-dependent gene expression. The time course of NF-B activation leading to d2EGFP expression was measured in an oligonucleotide-based NF-B-ELISA. NF-B binding in-creased after 15-min incubation with TNF-. In parallel, d2EGFP increased after 3 h and reached its maximum at 24 h. These results show (1) the time lag between NF-B activation and d2EGFP transcription, translation, and protein folding and (2) the increased reporter gene expression after treatment with TNF- to be caused by the activation of NF-B. The detection of d2EGFP expression required FACS analysis or fluorescence microscopy, while EGFP could also be measured in the microplate reader, rendering the assay useful for high-throughput screening. (Journal of Biomolecular Screening 2003:511-521)

Key Words: Nuclear Factor B • tumor necrosis factor • bioassays • reporter genes • green fluorescent protein • mammalian cells • transfection

Journal of Biomolecular Screening, Vol. 8, No. 5, 511-521 (2003)
DOI: 10.1177/1087057103257204


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